It is estimated that 1.2 million infants die annually from birth asphyxia and its complication, hypoxic- ischemic (HI) brain injury. In U.S. neonatal HI-brain injury remains one of the major causes for a life-long neurological disability in children. This indicates an urgent need to develop therapeutic strategies based on a better understanding the mechanisms of HI injury in the developing brain. Mitochondrial dysfunction is the most fundamental biological event leading to neuronal injury in this disease. Although, mitochondrial complex-I (C-I) is severely inhibited by a HI insult, upon re-oxygenation/reperfusion the energy-generating function of C-I rapidly recovers. This reperfusion-driven re-activation of C-I is tightly linked to the generation of reactive oxygen species (ROS). We hypothesize that the inhibition of mitochondrial C-I recovery upon reperfusion represents a therapeutic strategy against an oxidative burst during early reperfusion. We show that compared to controls, mice exposed to C-I inhibitor, pyridaben exhibited significant attenuation of cerebral injury, despite a sluggish recovery of the C-I linked mitochondrial respiration. Mitochondria isolated from these pyridaben-exposed HI-mice demonstrated a limited acceleration in ROS production during reperfusion. This suggests that during reperfusion post-HI restoration of electron transport flow in the C-I contributes not only to cellular recovery, but also to cellular injury. Specific aims are designed to determine; Aim 1, whether an inhibition of the C-I recovery during reperfusion attenuates oxidative damage to mitochondrial matrix and as a result, increases mitochondrial tolerance to Ca++ induced opening of permeability transition pore (PTP). and Aim 2, whether hypoxemia during reperfusion slow down reactivation of mitochondrial C-I and whether this protects brain against reperfusion-driven oxidative stress Thus, the project is designed to establish both, mechanistic rationale and a clinical translation for an innovative therapeutic concept of a gradual metabolic recovery of the C-I directed against reperfusion-driven oxidative stress. PUBLIC HEALTH RELEVANCE: This proposal is focused to develop a novel therapeutic concept for protection of the ischemic brain against reperfusion-driven oxidative stress. Potential implication of this research is very broad, because an ischemic brain injury (stroke, hypoxic-ischemic encephalopathy) is a leading cause for neurological handicap in adults and children.